Expression of feline leukaemia virus antigens on cat lymphoma cells: kinetics of biosynthesis.
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Biomedical subjects
Publications and source records attributed to S J O'Brien.
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Somatic cell hybrids were constructed between BALB/c-RAG mouse cells and feline lymphoma cells by the hypoxanthine-aminopterin-thymidine selection scheme. RAG cells spontaneously produce an endogenous B-tropic type C virus. Cat-mouse hybrids preferentially segregate feline chromosomes and retain murine chromosomes-demonstrable by karyotypic and isozyme analyses. Despite the presence of the complete mouse genome, including the viral genome, virus production was diminished to 1-5% of the levels observed in RAG parents based upon particle-associated RNA-dependent DNA polymerase (reverse transcriptase) activity in the culture fluid. Thirty-seven hybrids made on four different occasions had suppressed virus levels, and no hybrids expressed parental virus levels. Reverse selection experiments on 6-thioguanine demonstrated that a restriction gene, tentatively named Bvr-1, was linked to the feline structural genes for hypoxanthine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase; EC 2.4.4.8) and glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase; EC 1.1.1.49) in cats, probably on the X-chromosome. The genetic mode of action of Bvr-1 is trans dominant in restriction of murine leukemia virus. The restriction locus results in a block late in virus maturation but prior to release, since expression of antigens for viral structural proteins and matrue budding particles is apparent on surfaces of restriced hybrid cells but not in high-speed pellets from culture fluid of restricted cells.
We have used a quantitative radiolabelled antibody procedure to measure the amount of certain virus structural antigens on the surface of BALB/c RAG cells producing endogenous B-tropic type C virus. RAG cells expressed group specificities of MuLV p30 on their cell surface but did not express gp70 group specificities. However, type specificities of gp70 were expressed on BALB/c cell lines infected with Moloney leukaemia virus. The majority of p30 antigens detected on the RAG cell surface were removed by trypsin and their reappearance was prevented by cycloheximide, even in the presence of 'conditioned medium' containing MuLV. Passive adsorption of exogenous MuLV p30 to the surface of virus negative BALB/c fibroblasts reached a maximum of 20% of the protein detectable on virus producing RAG cells. These data support the hypothesis that much, but not all, of the surface p30 is expressed de novo on the cell membrane and not derived from passive adsorption of p30 released from shed virus or as a by-product of virus infection of a cell.
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"Null" mutations previously isolated at the alphaGpdh-1 locus of Drosophila melanogaster, because of disruption of the energy-producing alpha-glycerophosphate cycle, severely restrict the flight ability and relative viability of affected individuals. Two "null" alleles, alphaGpdh-1(BO-1-4), and alphaGpdh-1(BO-1-5,) when made hemizygous with a deficiency of the alphaGpdh-1 locus, Df(2L)GdhA, were rendered homozygous by recombination with and selective elimination of the Df(2L)GdhA chromosome. After over 25 generations, a homozygous alphaGpdh-1(BO-1-4) stock regained the ability to fly despite the continued absence of measurable alphaGPDH activity. Inter se heterozygotes of three noncomplementing alphaGpdh-1 "null" alleles and the "adapted" alphaGpdh-1(BO-1-4) homozygotes were examined for metabolic enzymatic activities related to the energy-producing and pyridine nucleotide-regulating functions of the alpha-glycerophosphate cycle in Drosophila. The enzyme functions tested included glyceraldehyde-3-phosphate dehydrogenase, cytoplasmic and soluble malate dehydrogenase, lactate dehydrogenase, mitochondrial NADH oxidation, oxidative phosphorylation, and respiratory control with the substrates alpha-glycerophosphate, succinate, and pyruvate. These activities in any of the mutant genotypes in early adult life were indistinguishable from those in the wild type. There was, however, a premature deterioration and atrophy of the ultrastructural integrity of flight muscle sarcosomes observed by electron microscopy in the "null" mutants. These observations were correlated with a decrease in state 3 mitochondrial oxidation with alpha-glycerophosphate, succinate, and pyruvate, as well as with loss of respiratory control in adults as early as 2 wk after eclosion. Such observations, which normally are seen in aged dipterans, were accompanied by premature mortality of the mutant heterozygotes. The adapted alphaGpdh-1(BO-1-4) was identical with wild type in each of the aging characters with the single exception of lowered rates of mitochondrial oxidative phosphorylation.
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A method for detecting possible structural genes in D. melanogaster based on gene dosage dependency is presented. By making thirty crosses between Y-autosome translocations, and an attached-4 cross, it is possible to produce large duplications (approximately 150 salivary gland chromosome bands in length) for every autosomal region with the exception of 83DE. The usefulness of the technique was demonstrated by dosage dependency of three known gene-enzyme systems: alpha-glycerophosphate dehydrogenase-1, alcohol dehydrogenase and malate dehydrogenase. A screen for genes affecting two enzymes localized on the inner membrane of the mitochondrion, alpha-glycerophosphate oxidase (alphaGPO) and succinic dehydrogenase (SHD), produced a dosage-sensitive region in each case. Region 50C-52E affected alphaGPO activity and region 28D-29F affected SDH activity. The latter region apparently includes the malic dehydrogenase-1 gene. The methodology and limitations of the technique are discussed.
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Seven alleles of the alpha-Glycerophosphate dehydrogenase-1 (alphaGpdh-1) locus of Drosophila melanogaster have been described. These include two naturally occurring electrophoretic variants, one EMS-induced electrophoretic variant, and four EMS-induced "null" or "zero" mutants. With the electrophoretic variants, the locus was mapped to II-20.5 +/- 2.5. A complementation matrix was prepared utilizing the null mutants. Three of the four mutants and a deletion of the locus (Grell 1967) exhibit dosage dependency. The dosage independent mutant exhibits complementation with two of the other null alleles. Flies genetically deficient in alpha-glycerophosphate dehydrogenase are fertile, but their relative viability is severely diminished. Such flies also lose the ability to sustain flight, an observation consistent with the enzyme's function in energy production. The levels of mitochondrial alpha-glycerophosphate oxidase, measured in flies genetically deficient in the cytoplasmic enzyme, were normal.
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Presented here are the detailed methods employed in our laboratory for gene mapping and cytogenetic analyses in human beings, in the domestic cat, and in other mammalian species. Induced in the procedures are: 1) establishment of primary fibroblast and lymphoid cell cultures; 2) heterologous cell fusion for production of rapidly proliferating cell hybrids; 3) cellular transformation of primary fibroblasts using an oncogenic retrovirus; 4) cell synchronization for high-resolution banding of promethaphase chromosomes; 5) chromosome-banding procedures, including G-banding, alkaline G-11, and Q-banding; and 6) in situ hybridization of radiolabeled molecular clones to metaphase chromosomes for regional gene localization.
Nine patients had combined anterior cruciate ligament (ACL) disruption and patella dislocation and underwent surgical reconstruction of one or both of these injuries. Six patients had both the ACL reconstructed and the patella realigned, and three had only the ACL reconstructed. Associated injuries were present in eight cases; these included meniscal tears in eight patients and medial collateral ligament injuries in two of these same patients. At final follow-up, at an average 19.7 months, examination revealed an average grade 1A Lachman and no pivot on all patients who underwent ACL reconstruction. No patients had hypermobile patellae or apprehension. One patient had a 4 degrees loss of extension and none had a loss of flexion. Two patients had continued anterior knee pain at final follow-up; one of these patients was the same person who had a loss of extension. None had recurrent instability of the ACL and none had recurrent instability of the patella.
We performed a retrospective review to evaluate acute medial collateral ligament injuries of the elbow in professional football players from 1991 to 1996 (5 seasons). There were 5 acute medial collateral ligament injuries in 4 players (1 player with bilateral involvement). All injuries occurred with the hand planted on the playing surface while a valgus or hyperextension force was applied to the elbow. There were 2 centers, both involved with long-snapping situations, 1 running back, and 1 quarterback. All elbows had valgus instability on physical examination. Despite this instability, all players were able to function without operative reconstruction of the medial collateral ligament. No evidence of valgus instability was seen at the time of follow-up (average, 3.4 years). Next, we reviewed all acute elbow injuries in the National Football League from the same 5-season period. Ninety-one acute elbow injuries were reviewed. Overall, there were 70 (76.9%) elbow sprains, 16 (17.6%) dislocation/subluxation patterns, 4 (4.4%) fractures, and 1 (1.1%) miscellaneous injury. Review of the acute elbow sprains revealed 39 (55.7%) hyperextension injuries, 14 (20%) medial collateral ligament injuries, 2 (2.9%) lateral collateral ligament sprains, and 15 (21.4%) nonspecific sprains. The epidemiology of the 14 medial collateral ligament injuries was studied in more detail. The 2 most common mechanisms of injury were blocking at the line of scrimmage (50%) and the application of a valgus force with the hand planted on the playing surface (29%). There were 8 linemen, 4 receivers, 1 running back, and 1 quarterback. All injuries were managed with nonoperative treatment. The average time lost was 0.64 games (range, 0 to 4). We report 19 acute medial collateral ligament injuries of the elbow in elite football players, 2 of whom are considered overhead throwing athletes, who were able to function at a competitive level without surgical repair or reconstruction, in contrast to baseball players, in whom the mechanics and demands may differ.
Twenty-three fresh-frozen cadaver shoulders free of degenerative arthritis or rotator cuff disease were tested biomechanically to quantitate the contribution of specific capsular structures to restricting anterior-posterior translation of the abducted shoulder. With the glenohumeral joint in 90 degrees of abduction on a servohydraulic control testing system, translation was measured in 30 degrees of forward flexion (with regard to the coronal plane of the scapula), 0 degree, and 30 degrees of extension while a 25 N anterior-posterior load was applied. Measurements were taken both in the intact (vented) shoulder and after selective cutting of different capsuloligamentous structures was performed. In the intact shoulder the largest anterior-posterior translation occurred in 0 degree of horizontal flexion and extension with regard to the scapular plane, with equal amounts of anterior and posterior translation noted. The primary anterior-posterior stabilizer of the abducted shoulder is the inferior glenohumeral ligament complex. The anterior band is the primary stabilizer in 30 degrees of horizontal extension and at 0 degree (neutral). The posterior band is the primary stabilizer in 30 degrees of horizontal flexion. This study quantifies for the first time the normal amount of anterior-posterior translation in the intact cadaveric shoulder model. In addition, it demonstrates the relative role of the anterior and posterior band of the inferior glenohumeral ligament complex in stabilizing the glenohumeral joint at 90 degrees of abduction, where most clinical instability of the shoulder occurs.
Ten cadaveric shoulders were tested to evaluate the effect of simulated contraction of the long head of the biceps brachii on glenohumeral translation. The shoulders were mounted on a special apparatus attached to a servo-controlled hydraulic testing device. Sequential 50 N anterior, posterior, superior, and inferior forces and a 22 N joint compressive load were applied to the shoulders. An air cylinder applied a constant force to the tendon of the long head of the biceps brachii. The shoulders were tested in seven positions of glenohumeral elevation and rotation. Application of a force to the long head of the biceps brachii resulted in statistically significant decreases in humeral head translation. The influence of the long head of the biceps was more pronounced at middle and lower elevation angles. When the shoulder was placed in 45 degrees of elevation and neutral rotation, application of a 55 N force to the biceps tendon reduced anterior translation by 10.4 mm (p = 0.001), inferior translation by 5.3 mm (p = 0.01), and superior translation by 1.2 mm (p = 0.004).
Shoulder capsular shrinkage has recently been proposed as a therapeutic modality in a select group of patients with instability. Basic science research studying the mechanism of collagen shrinkage and the effect of shrinkage on the tissue's mechanical properties is essential to define the ideal process by which to achieve optimal tissue shrinkage. Tissue shrinkage is a function of both time and temperature. This relationship was studied, and a model was derived to describe the relationship mathematically. Tissue shrinkage rate was extremely sensitive to temperature changes. The purpose of this study, was to shrink collagenous tissue thermally and then to measure the mechanical property changes as a function of tissue shrinkage. Uniaxial tensile testing of normal and heat-shrunken bovine tendon was carried out, and a model was developed to express the relationship between shrinkage and mechanical properties. We found that the mechanical properties decreased with increasing shrinkage, and that the maximal allowable shrinkage before significant material property changes occurred was between 15% to 20%. Ultrastructural analysis with transmission electron microscopy showed denaturation of the collagen fibrillar structure and provided direct support for the observed material changes.
The application of recombinant DNA technologies has allowed the detection of at least three families of moderately repetitive DNA segments in the human genome that are homologous to retroviruses previously isolated from mice and primates. One of these DNA segments has been shown by nucleotide sequence comparisons to be distantly related to both Moloney murine leukaemia virus (MoMuLV) and the endogenous baboon retrovirus and to have the sequence organization characteristic of an integrated retrovirus. Isolation of the homologous locus from chimpanzee DNA indicated that the integration event preceded the evolutionary divergence of chimpanzees and man. Here we have used a panel of rodent x human somatic cell hybrids to assign the chromosomal localization of this segment, called ERV1 (endogenous retrovirus-1), to human chromosome 18 (HSA 18).